Angiotensin-(1-7)Administration Attenuates Alzheimer’s Disease-like Neuropathology In Rats With Streptozotocin-induced Diabetes Via Mas Receptor Activation

Objectives1.To investigate the effect of angiotensin-(1-7)[Ang-(1-7)] on cognitive function in rats with streptozotocin(STZ)induced diabetes.2.To investigate the effect of Ang-(1-7)on brain ACE2/Ang-(1-7)/Mas axis and the role of Mas receptor.3.To investigate the effect of Ang-(1-7)on synaptic structures in hippocampal CA1 neurons,the phosphorylation of Tau protein and the β-amyloid proteins(Aβ).Methods1.Sprague-Dawley male rats were randomly divided into four groups.Control group,diabetic group(DM group),Ang-(1-7)group and Ang-(1-7)+A779 group.Diabetes was induced via single i.p.streptozotocin(STZ)injections(55 mg/kg).Ten weeks after diabetes induction,rats in each group received an intracerebral-ventricular(ICV)infusion of either vehicle,Ang-(1-7)alone,or Ang-(1-7)+A779 daily for 11 two weeks.2.Morris water maze(MWM)tests were performed to test cognitive functions before the rats were euthanized.3.Immunohistochemistry(IHC)and western blot were used to test the ACE2/Ang-(1-7)/Mas axis in the hippocampi of rats.4.Transmission electron microscopy(TEM)was used to detect the ultrastructure of synapses in hippocampal CA1 area.IHC and western blot were used to detect the hyperphosphorylated Tau and Aβ oligomers,ELISA to detect the soluble and insoluble Aβ1-40 and Aβ1-42 in the hippocampi of rats.Results1.DM group showed significantly prolonged escape latencies than the control group,Ang-(1-7)treatment significantly reduced escape latencies in diabetic rats in acquisition trials.In probe trials,DM group reduced platform area crossing frequency and time spent in the target quadrant compared with the control group,and Ang-(1-7)group markedly enhanced platform area crossing frequency and time spent in the target quadrant(3.0±0.39 versus 1.0±0.33,39.39±1.11% versus 25.62±3.07%,respectively,P<0.01).2.The expression of ACE2 and Mas receptor were markedly reduced in the hippocampi of DM group compared with the control group,Ang-(1-7)treatment significantly enhanced the expression of ACE2 and Mas receptor in the hippocampus.3.TEM showed that Ang-(1-7)treatment ameliorated damage to the ultrastructure of hippocampal synapses.IHC and western blot showed that Ang-(1-7)reduced the expression of hippocampal phospho-tau at Ser396(P<0.01),Ser404(P<0.01)and Ser202/Thr205(P<0.05).ELISA and IHC showed decreased both soluble and insoluble β-amyloid peptide 1-42(Aβ 1-42)and Aβ 1-40 levels(P<0.01)with the treatment of Ang-(1-7),reduced the formation of Aβ1-42 plaques.Western blot and IHC showed that Ang-(1-7)reduced the Aβ oligomers.4.These neuro protective effects were significantly reversed by the co-administration of A779.ConclusionsThese findings show that Ang-(1-7)can improve the cognitive function in diabetic rats and can attenuate AD like neuropathology in the hippocampi of diabetic rats.Ang-(1-7)is a promising therapeutic target for diabetes-induced cognitive impairment.The neuroprotective effects of Ang-(1-7)were mainly through Mas receptor(Mas R)activation.Objectives1.To investigate the effect of metformin on cognitive function in db/db mice.2.To investigate the effect of metformin on phosphorylated Tau proteins and autophagic activity in db/db mice hippocampi and high-glucose induced HT22 cells.And further investigate the potential molecular mechanisms on how metformin affects the phosphorylated Tau proteins and autophagic activity.Methods1.db/db mice were randomly divided into four groups: db/db group,metformin group(Met group),metformin+chloroquine group(Met+CQ group)and chloroquine group(CQ group),db/+ mice were used as control group.Met group,Met+CQ group and CQ group received i.p.injections of metformin(200mg/kg/d),metformin(200mg/kg/d)+ chloroquine(10mg/kg/d)and chloroquine(10mg/kg/d),respectively,for consecutive eight weeks.Control group and db/db group received equivalent volume of saline.High glucose induced HT22 cells was used as the in vitro model of diabetes,normal glucose incubated HT22 cells was used as control.2.Morris water maze(MWM)tests were performed to test cognitive functions before the mice were euthanized.3.Immunohistochemistry(IHC),Immunofluorescence(IF)and western blot were used to test the effect of metformin on expression of phosphorylated Tau proteins both in vivo and in vitro.4.Transmission electron microscopy(TEM)and Western blot were used to detect the effect of metformin on autophagy both in vivo and in vitro.We further used CQ(in vivo)and 3-MA(in vitro)as autophagic inhibitors to investigate whether metformin attenuates phosphorylated tau proteins via modulating autophagic clearance.5.Compound C was used as AMPK inhibitor in vitro.Western blot and TEM were used to detect whether metformin modulates autophagy through AMPK activation.Results1.Metformin treatment significantly reduced escape latencies in diabetic mice in acquisition trials.Met+CQ group showed significantly prolonged escape latencies than the Met group(P<0.05).In probe trials,Met group enhanced platform area crossing frequency and time spent in the target quadrant compared with db/db group(P<0.05),and Met+CQ group markedly reduced platform area crossing frequency and time spent in the target quadrant(P<0.05).2.Metformin treatment significantly reduced the expression of p-Tau(Ser396),p-Tau(Ser404),AT8(Ser202/Thr205)in db/db mice hippocampi and high glucose induced HT22 cells(P<0.01).3.Metformin enhanced autophagy activity in db/db mice hippocampi and high glucose induced HT22 cells.The effect of metformin attenuated p-Tau(Ser396),p-Tau(Ser404),AT8(Ser202/Thr205)expression in db/db mice hippocampi and high glucose induced HT22 cells was significantly reversed by inhibiting the autophagic flux in vivo and in vitro(P<0.01).4.Metformin treatment significantly enhanced AMPK activity in db/db mice hippocampi and high glucose induced HT22 cells(P<0.01).The effect metformin induced autophagy in high glucose induced HT22 cells was reversed by Compound C.ConclusionsThese findings show that metformin can improve the cognitive function in db/db mice and can attenuate tau hyperphosphorylation in the hippocampi of db/db mice and in high glucose induced HT22 cells.These neuroprotective effects are mainly through enhancing autophagic clearance via activation of AMPK.